Lipobeads and their production

ABSTRACT

Lipobeads (liposome-encapsulated hydrogels) combine properties of hydrogels and liposomes to create systems that are sensitive to environmental conditions and respond to changes in those conditions in a fast time scale. Lipobeads may be produced by polymerizing anchored or unanchored hydrogels within liposomes or by mixing anchored or unanchored hydrogels with liposomes. Giant lipobeads may be produced by shrinking unanchored nanogels in lipobeads and fusing the resulting lipobead aggregates, long-term aging of anchored or unanchored lipobeads, or mixing anchored or unanchored aggregated nanogels with liposomes. Poly(acrylamide), poly(N-isopropylacrylamide), and poly(N-isopropylacrylamide-co-1-vinylimidazole) lipobeads were produced and characterized.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/218,553 (referred to as “the '553 application” and incorporatedherein by references), titled “LIPOBEADS AND THEIR PRODUCTION,” filed onAug. 14, 2002, listing Sergey Kazakov, Marian Kaholek, and Kalle Levonas the inventors, scheduled to issue as U.S. Pat. No. 7,618,565 on Nov.17, 2009, and claiming benefit, under 35 U.S.C. §119(e) (1), to thefiling date of provisional patent application Ser. No. 60/312,878(referred to as “the '878 provisional” and incorporated herein byreference), entitled “UV-INDUCED GELATION ON NANOMETER SCALE USINGLIPOSOME REACTOR”, filed on Aug. 16, 2001 and listing Sergey Kazakov,Marian Kaholek, and Kalle Levon as the inventors, for any inventionsdisclosed in the manner provided by 35 U.S.C. §112, ¶1.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government support and the Government hascertain rights in the invention as provided for by contract number0660076225 awarded by DARPA.

BACKGROUND

1. Field of the Invention

The present invention concerns nanogels encapsulated within a lipidbilayer (lipobeads) and their production.

2. Related Art

Artificial systems consisting of only spherical hydrogel particles orliposomes have already found a variety of biomedical applications indrug delivery, drug targeting, protein separation, enzyme immobilizationand so on. Sensory properties of the combined liposome-hydrogelstructures may lead to novel biomimetic sensory systems.

Liposomes are phospholipid assemblies consisting of a flexible, cellmembrane-like lipid bilayer, the surface of which acts as a permeabilitybarrier. Different compounds can be entrapped in the liposome's aqueousinterior. It has been shown that liposomes can be constructed withbilayer permeability responsive to a variety of physical and chemicalstimuli, including temperature, light, pH, and ions (See, e.g., G.Gregoriadis et al., Vesicles, Marcel Dekker: New York (1996). This bookis incorporated herein by reference.). These liposomes can mimic variousfunctions of biological membranes and can be used as a container forstorage, transport, and controllable release of compounds. Liposomes canbe mechanically unstable, however and their loading capacity is limitedby the water solubility of the material to be loaded.

Hydrogel particles are mechanically more stable than liposomes becauseof cross-linking and have larger loading capacities than liposomes.Their properties (swelling/de-swelling) can be more sensitive toenvironmental conditions. It has been reported that some polymer gelscan swell or shrink discontinuously and reversibly in response to manydifferent stimuli (temperature, pH, ions, electric fields or light)depending on the chemical composition of the gel/solvent system. Thevolume change can be as large as a thousand-fold. Macroscopic gelsrespond to the environmental changes on a rather long-time scale,however. (See, e.g., the article Tanaka et al., J. Chem. Phys., 90: 5161(1989). This article is incorporated herein by reference.) The Tanakaarticle showed that for a spherical gel, the time required for swellingor shrinking is proportional to the square of its radius. Therefore,smaller hydrogels should swell/deswell faster. Such smaller hydrogels(e.g., having a diameter on a nanometer scale) should find morepotential applications.

Nevertheless, hydrogels lack many useful surface properties of a lipidbilayer. Lipid bilayers stabilized on various supports (glass, plastic,metal, and modified polymer) (See, e.g., the articles: Bayer et al.,Biophys. J., 58: 357 (1990); Rothe et al., FEBS. Lett., 263: 308 (1990);Plant, Langmuir, 9: 2764 (1993); Spinke et al., Biophys. J., 63: 1667(1992). These articles are incorporated herein by reference.) have founda number of applications (See, e.g., the articles: Sackman, Science,271: 43 (1996); McConnell et al., Biochim. Biophys. Acta, 864: 95(1986). These articles are incorporated herein by reference.). Bilayermembranes on solid supports are attractive systems mimicking thestructural, sensing, and transport roles of biological membranes (See,e.g., the articles: Woodhouse et al., Faraday Discuss, 111: 247 (1998);Wagner et al., Biophys. J., 79: 1400 (2000); Raguse et al., Langmuir,14: 648 (1998); Cornell et al., Nature, 387: 580 (1997); Kasianowicz etal., Anal. Chem., 73: 2268 (2001). These articles are incorporatedherein by reference.), especially sensory systems using ion-channelswitches (See, e.g., the articles Raguse et al., Cornell et al., andKasianowicz et al.). The main drawback of the supported bilayermembranes to date is a lack of well-defined ionic reservoirs on bothsides of the membrane.

A functional ionic reservoir between membrane and a substrate can beconstructed using an ion sensitive hydrogel. In this context,combination of hydrogel particles with liposomes reconstituted with themembrane protein (ionic channel) can be considered as a model system tostudy the functions of membranes and membrane proteins and to design newsensory devices. An appropriate assembly of lipid bilayer on a sphericalhydrogel surface can be used to prepare an artificial cell analogue.Furthermore, hydrogel-liposome assemblies combine the properties of bothclasses of materials, which broaden their potentials for biomimeticsensory systems, controlled release devices, and multivalent receptors.

Work on preparing and characterizing submicrometer-scale hydrogelparticles has intensified recently, but there are few works devoted tofabricating different combinations of hydrogels and liposomes. A methodof fabricating hydrogel spherical particles (beads) within liposomes wasreported (See U.S. Pat. No. 5,626,870, hereafter referred to as “theMonshipouri patent”; V. P. Torchilin et al., Macromol. Chem., RapidCommun. 8: 457 (1987), hereafter referred to as “the Torchilin article”.These works are incorporated herein by reference.). Unfortunately,however, the method discussed in the Monshipouri patent required specialhydrogel-forming substances with a gelation initiator for which aliposomal bilayer was permeable. The authors of the Torchilin articleprepared LUV liposomes with average diameters of approximately 650 nmusing the reverse phase evaporation method. This technique, however,makes it difficult to control liposome size and polydispersity, which isthe reason that the Torchilin article presents only average sizes of theparticles detected by dynamic light scattering. According to theTorchilin article, the detergent and phospholipid were not removed aftersolubilizing the lipid bilayer to release the hydrogel particles.Moreover, gels contained in liposomes and gel particles were notdistinguished by scanning electron microscopy. Encapsulating hydrogelparticles in liposomes was described (See, e.g., the article, Gao, K.;Huang L. Biochim. Biophys. Acta. 897: 377 (1987), hereafter referred toas “the Gao article”. This article is incorporated herein byreference.). Although the overall mechanical strength of the liposomalstructure discussed in the Gao article was enhanced in the lattersystem, the unanchored bilayer was still unstable and needed specificlipid mixtures and polymer cores of certain sizes and shapes. Thearticle by Jin et al. (FEBS Lett. 397: 70 (1996). This article isincorporated herein by reference.) reported the design and preparationof a novel hydrogel-anchored lipid vesicle system, named “lipobeads”.This system contained (i) a hydrogel polymer core anchored by fattyacids, which were covalently attached to the surface of the hydrogel and(ii) a lipid monolayer around the modified hydrogel spherical particle.In this system, the bilayer consisting of hydrophobic chains of fattyacids and hydrophobic tails of the phospholipids, was more stable thanthat in the system discussed in the Gao article. Spherical anionicmicrogels (6.5 μm at pH 7.0), composed of methylene-bis-acrylamide andmethacrylic acid and loaded with doxorubicin, were coated with a lipidbilayer (See Kiser et al., Nature, 394: 459 (1998). This article isincorporated herein by reference.) to control swelling and release ofdoxorubicin from the microgels. (See, e.g., the article Yang et al., J.Chromotogr. B. 707: 131 (1998). This article is incorporated herein byreference.) Biotinylated small and large unilamellar liposomes wereimmobilized in avidin- or streptavidin-derived gel beads forchromatographic analysis. Recently, it was reported that eggphosphatidylcholine liposomes and biomembrane fragments could beimmobilized on the surface of poly(acrylamide) macrogel containinghydrophobic anchors, which probably penetrated into the lipid bilayer(See, e.g., the article Yang, et al. Mat. Sci. and Eng. C. 13: 117(2000)). In all of the above-referenced works (except the Monshipouripatent and the Torchilin article), the sizes of hydrogel particlesvaried on the micrometer scale. (In these works, optical or electronmicroscopy was used for characterization.) However, hydrogel particleswith nanometer-range diameters would swell and shrink faster in responseto environmental conditions because of their smaller radii.

In view of the limits of the state of the art, hydrogel/liposome systemsthat can respond to changes in the environment on a short time scale areneeded.

SUMMARY OF THE INVENTION

The present invention describes preparing and characterizing hydrogelnanoparticles (nanogels) and liposomes. The present invention alsodescribes different assemblies of nanogels and liposomes definingvarious hydrogel/liposome systems. These hydrogel/liposome systems willoften combine complementary advantages of the liposomes and thepolymeric hydrogels. Studying the individual behavior of the hydrogelparticles and liposomes in aqueous solution affords better understandingof the behavior of the hydrogel/liposome system. The following systemswere prepared and characterized: (i) an unanchored nanogel entrapped ina liposome; (ii) an anchored nanogel entrapped in a liposome; (iii) anaggregate of unanchored nanogels coated with phospholipid bilayer(“giant” lipobeads), (iv) an aggregate of anchored nanogels coated withphospholipid bilayer (“giant” lipobeads), (v) an aggregate of anchoredand unanchored nanogels coated with phospholipid bilayer (combined giantlipobeads) and (vi) an aggregate of anchored lipobeads.

The present invention also describes the size distribution changes ofthe hydrogel particles, liposomes, and their assemblies in response tosolvent variations, temperature variations, pH variations, and ionicstrength variations.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a scheme depicting lipobead preparation by polymerizinghydrogel-forming components within liposomes.

FIG. 2 is a scheme depicting lipobead preparation by constructingliposomes and nanogels independently and then mixing them together.

FIG. 3 shows graphs of the size distribution curves for (a) liposomes,(b) pure PNIPA nanogels, and their mixture below (c) and above (d) thevolume phase transition temperature.

FIG. 4 shows graphs of the size distribution curves for (a) liposomes,(b) anchored PNIPA-VI nanogels, and their mixture (c) below and (d)above the volume phase transition temperature.

FIG. 5 is a tapping mode Atomic Force Microscopy (AFM) image (amplitudedata) of the anchored PNIPA-VI nanogel (1) coated by a lipid bilayer (2)obtained after mixing PNIPA-VI nanogels with liposomes at 25° C.(bar=100 nm).

FIGS. 6 a and 6 b are AFM images (amplitude data) of (a) a PNIPA-VIlipobead after gelation inside liposomes (frame 400×400 nm²; 1,flattened lipid bilayer; 2, nanogel); and (b) mixedphospholipid/detergent micelles and a nanogel after lipid bilayersolubilization (frame 440×440 nm²; 1, nanogel; 2, mixed micelles).

FIG. 7 shows graphs of the size distribution curves for (a) PNIPA-VIlipobeads in buffer (pH 7.5) and after addition of 15 mM T_(X-100) at(b) pH 7.5 and (c) pH 2.5.

FIG. 8 shows a graph of the size distribution curve for PAAm lipobeadsin buffer.

FIGS. 9 a and 9 b are (a) an AFM images (amplitude data) of giantlipobeads produced by long-term aging of a mixture of EPC liposomes andPNIPA-VI nanogels after three months (frame 10×10 μm²) and (b) aschematic presentation of the giant lipobeads formation in aging.

FIG. 10 shows graphs of (a) the Z-average diameter of liposomes as afunction of temperature during heating/cooling cycles and (b) pHDependence of the average diameter of the liposomes.

FIG. 11 shows graphs of the size distribution curves for the pure PNIPAhydrogel particles in water (a) below and (b) above the volume phasetransition temperature.

FIG. 12 shows graphs of the size distribution curves for (a) PNIPAlipobeads in buffer at 25° C., (b) after addition of 15 mM T_(X-100) at25° C., (c) at 35° C., and (d) at 40° C.

FIG. 13 is a table that lists the composition of hydrogel-forming mediaand properties of macroscopic hydrogels.

FIG. 14 is a graph of the Z-average diameter of anchored PNIPA-VIlipobeads (a) as a function of temperature upon heating/cooling cyclesand (b) as a function of pH.

FIG. 15 includes graphs showing the effect of temperature on thecollapse and subsequent aggregation of PNIPA-VI nanogels for purePNIPA-VI nanogels in water at temperatures below, and at a temperatureabove, the volume phase transition temperature.

FIG. 16 is a graph showing the pH dependence of the average diameter ofPNIPA-VI nanogels.

DETAILED DESCRIPTION

The present invention concerns lipobeads, giant lipobeads, theirproduction, and their properties. The present invention functions toproduce assemblies of liposome-encapsulated nanogel particles thatrespond to environmental conditions such as pH, temperature, and ions ona fast time scale. The following description is presented to one skilledin the art to make and use the invention, and is provided in the contextof particular embodiments and methods. Various modifications to thedisclosed embodiments and methods will be apparent to those skilled inthe art, and the general principles set forth below may be applied toother embodiments, methods and applications. Thus, the present inventionis not intended to be limited to the embodiments and methods shown andthe inventors regard their invention as the following disclosed methods,apparatus and materials and any other patentable subject matter to theextent that they are patentable.

In the following, fabrication of nanogels, liposomes, lipobeads, andgiant lipobeads is described in §4.1. Properties of the resultingliposomes, nanogels, lipobeads and giant lipobead systems are describedin §4.2 in terms of exemplary embodiments.

Generating Hydrogel/Liposome Assemblies

In the following, generating liposomes and nanogels are described in§4.1.1 and §4.1.2. Combinations of hydrogels and liposomes, lipobeadsand giant lipobeads, are described in §4.1.3 and §4.1.4.

Generating Liposomes

Liposome preparation is described in U.S. patent application Ser. No.10/218,554, filed concurrently with the '553 application on Aug. 14,2002, entitled NANOGELS AND THEIR PRODUCTION USING LIPOSOMES ASREACTORS, by Sergey Kazakov, Marian Kaholek, and Kalle Levon (Thispatent is incorporated herein by reference). Generally, this techniqueof preparing liposomes involves freezing and thawing a solution ofmultilamellar vesicles (MLV) followed by sonication to yield largeunilamellar vesicles (LUV) within the range of average sizes from 30 to1000 nm.

Generating Nanogels

Preparing hydrogels with nanometer-scale dimensions using liposomes asreactors was described in U.S. patent application Ser. No. 10/218,554,filed concurrently with the '553 application on Aug. 14, 2002, entitledNANOGELS AND THEIR PRODUCTION USING LIPOSOMES AS REACTORS, by SergeyKazakov, Marian Kaholek, and Kalle Levon. In general, this technique ofpreparing nanogels involves (i) encapsulating hydrogel-formingcomponents into the liposomes and (ii) polymerizing the encapsulatedhydrogel-forming components.

Generating Lipobeads

The present invention may be used to produce lipobeads by (a)polymerizing hydrogel-forming components within liposomes, or (b) mixingnanogel particles with liposomes to form lipid bilayer-coated hydrogelparticles.

The first method prepares liposomes before gelation and then polymerizeshydrogel-forming components inside the liposomes, as shown in FIG. 1.This method has the advantage of forming a more stable lipid bilayer.However, nanogels inside the liposomes are unable to undergo furthermodifying treatments such as loading, entrapment, or surfacefunctionalization. Such procedures should be planned in the course ofgelation instead. Lipobead fusion and formation of “giant” or combinedlipobeads is impossible using this method. Polymerizing anchored orunanchored hydrogel-forming components within liposomes was described inU.S. patent application Ser. No. 10/218,554, filed concurrently with the'553 application on Aug. 14, 2002, entitled NANOGELS AND THEIRPRODUCTION USING LIPOSOMES AS REACTORS, by Sergey Kazakov, MarianKaholek, and Kalle Levon

The second method prepares liposomes and nanogels independently, asshown in FIG. 2. Using this method, penetration by a nanogel into aliposome ruptures the liposome's lipid bilayer. However, the lipidbilayer on the surface of nanogels is strong and stable enough to maskthe nanogels' sensitivity to changes in external conditions. Forexample, the sensitivity of PNIPA-VI lipobeads to temperature and pH wasmasked due to their lipid bilayer coat (See FIG. 11). The advantage offabricating liposomes and nanogels independently is that nanogels can beloaded with different compartments, filled with different liquid media,or/and functionalized with specific ligands before they are coated by alipid bilayer. Using this method, lipobeads may fuse to form giantlipobeads.

Using the second method depicted in FIG. 2, lipobeads may be prepared byadding a liposome suspension to a solution of unanchored or anchoredhydrogel particles. Incubating this solution for 1 to 4 hours at atemperature above the phase transition temperature of the phospholipid(Phospholipids' phase transition temperatures typically range fromapproximately −16 to +74° C. For example, egg phosphatidylcholine (EPC)has T_(p)=−2° C.) and vortexing until the suspension starts to becomehomogeneous (5-10 min) results in forming lipid bilayer-coated hydrogelparticles.

In an exemplary embodiment of the present invention, unanchoredpoly(N-isopropylacrylamide) (PNIPA) hydrogel particles (formed from 5-10wt. % hydrogel-forming components) were mixed in a 1:1 ratio withliposomes (formed from 5-10 mg/mL phospholipid in water or buffer),incubated for 2 hours at room temperature and vortexed to generateunanchored PNIPA lipobeads.

FIGS. 3 a and 3 b show the size distribution curves for the pureliposomes and pure PNIPA nanogels with approximately the same averagediameter of approximately 180 nm. Adding liposomes to the suspension ofpure PNIPA nanogels and observing the sizes of the resulting structuresallows one to determine if lipobeads form or if the particles remainseparated. If both particles existed independently without interactions,one could expect that upon increasing temperature the hydrogel particleswould shrink, whereas liposomal sizes would not change, and two separatepeaks would be detected. If both particles formed aggregates, however,the resulting particles should be at least twice their initial averagesize, as was seen in FIGS. 4 d and 3 b.

DLS measurements of the PNIPA nanogel/liposome mixture detected a singlepeak (FIG. 3 c) with an average diameter slightly greater than diameterof the initial liposomes and the PNIPA nanogels at 25° C. (e.g., belowT_(V) for PNIPA gels). The single peak (FIG. 3 d) significantly shiftedtowards larger diameters at 40° C. (e.g., above T_(V) for PNIPA gels).This unexpected behavior of the hydrogel particles/liposomes mixture canbe explained only by assuming that, during mixing, the liposomes'phospholipid bilayer covers PNIPA nanogels resulting in lipobeads, asillustrated in FIG. 3 c. Apparently, this configuration is energeticallymore preferable over the others. Note that considerable hydrophobicityof the PNIPA gel might be hidden within the liposome.

In another exemplary embodiment of the present invention, anchoredPNIPA-VI lipobeads were generated by mixing PNIPA-VI nanogels (formedfrom 5-15 wt. % hydrogel-forming components) with liposomes (formed from5-10 mg/mL phospholipid in water or buffer) in a 1:1 ratio, incubatingthe mixture for 2 hours at room temperature and vortexing.

FIGS. 4 a and 4 b show the size distribution curves for liposomes andpure anchored PNIPA-VI nanogels with average diameters of approximately160 nm, as was seen with PNIPA nanogels and liposomes (See FIG. 3).Mixing the liposomes and the anchored PNIPA-VI nanogels resulted inlipobeads with the size distribution curve shown in FIG. 4 c. Thesethree curves imply that the size distribution of the lipobeads isdetermined by the size distribution of the nanogels. Similar to mixingunanchored PNIPA nanogels and liposomes, each anchored PNIPA-VI hydrogelparticle (1) is coated with a liposomes' phospholipid bilayer (2),forming a lipobead during mixing, as shown in FIG. 5.

Polymerizing PNIPA-VI nanogels within liposomes also, as per U.S. patentapplication Ser. No. 10/218,554, filed concurrently with the '553application on Aug. 14, 2002, entitled NANOGELS AND THEIR PRODUCTIONUSING LIPOSOMES AS REACTORS, by Sergey Kazakov, Marian Kaholek, andKalle Levon, generated anchored PNIPA-VI lipobeads. Introducingwater-insoluble N-octadecylacrylamide (ODAm) into the liposomal membraneon the step of the dry phospholipid film formation resulted in nanogelsanchored to their liposomal reactors. Hydrophobic residues penetratedinto the lipid bilayer during liposome formation, and hydrophilic headsof ODAm were covalently bound to the polar end groups of the PNIPA-VIchains on the surface of the network during UV-copolymerization. As aresult, anchored PNIPA-VI lipobeads (See FIG. 6 a) with the diameter ofaround 200 nm (See FIG. 7 a) were synthesized.

Similarly, poly(acrylamide) (PAAm) hydrogels were generated as describedin U.S. patent application Ser. No. 10/218,554, filed concurrently withthe '553 application on Aug. 14, 2002, entitled NANOGELS AND THEIRPRODUCTION USING LIPOSOMES AS REACTORS, by Sergey Kazakov, MarianKaholek, and Kalle Levon. FIG. 8 shows a typical particle sizedistribution and structure of the lipobeads obtained afterpolymerization within liposomal microreactors.

Generating Giant Lipobeads

“Giant” lipobeads may be produced when lipobeads aggregate quickly dueto collapsing the hydrogel particles or slowly due to long-term aging(See FIG. 2).

Combining liposomes with hydrogel particles to form lipobeads andproviding the environmental conditions under which the hydrogelparticles collapse and the lipobeads aggregate together produces giantlipobeads. Collapsing unanchored lipobeads and incubating for 1 to 4hours at a temperature exceeding the volume phase transition temperatureof the polymer results in the formation of lipid bilayer-coated hydrogelaggregates, or giant lipobeads.

Anchored nanogels also may shrink inside liposomes due to temperaturesabove T_(v) and partially aggregate, but the hydrophobic chains(anchors) on their surface stabilize the lipid bilayer and preventfusion, thus prohibiting giant lipobeads from forming.

However, lipobeads with anchored or unanchored nanogels may form giantlipobeads after long-term aging (2-3 months) at a temperature below thevolume phase transition temperature of the polymer. (Lipobeads remainstable for approximately 1 month because the hydrogel stabilizes thelipid bilayer.) This method of producing giant lipobeads does notinvolve shrinking the nanogels inside the lipobeads.

In an exemplary embodiment of the present invention, unanchored hydrogelparticles are mixed with liposomes. Incubating the mixture for 2 hoursat a temperature above the volume phase transition temperature (32° C.for PNIPA nanogels) forms lipobeads. Heating to 40° C. causes thenanogels to collapse inside the lipobeads, reducing their totalhydrophobic surface area and resulting in lipobead aggregation. Afterincubating the mixture for 20 minutes at elevated temperatures, theaggregated lipobeads' lipid bilayers fuse to yield giant lipobeads witha structure depicted in FIG. 3 d for PNIPA lipobeads.

In another exemplary embodiment of the present invention, PNIPA andPNIPA-VI lipobeads aggregate and form giant lipobeads at roomtemperature (i.e., below volume phase transition temperature) withoutshrinking the nanogels inside the lipobeads. Aggregation and giantlipobeads formation occur over the course of long-term aging (2-3months) of the lipobeads. An AFM image (amplitude data) of anchoredPNIPA-VI giant lipobeads after three months storage at +4° C. ispresented in FIG. 9.

Properties of Systems Generated

One goal of the present invention is to produce a system that mimicsbiological entities and responds quickly to changes in environmentalconditions such as pH, ions, and temperature. Properties of liposomesand nanogels are described in §4.2.1 and §4.2.2, respectively.Properties of lipobeads and giant lipobeads are described in §4.2.3 and§4.2.4, respectively.

Properties of Liposomes

U.S. patent application Ser. No. 10/218,554, filed concurrently with the'553 application on Aug. 14, 2002, entitled NANOGELS AND THEIRPRODUCTION USING LIPOSOMES AS REACTORS, by Sergey Kazakov, MarianKaholek, and Kalle Levon showed that LUV liposomes had a sizedistribution between 30 and 1000 nm.

The temperature dependence of the apparent diameter of eggphosphatidylcholine (EPC) liposomes between 20 and 40° C. is shown inFIG. 10 a. In the temperature range above the main transitiontemperature (−2° C.), there was a slight decrease in <d> with anincreasing temperature. The decrease was less than 5% in the rangestudied. There was almost no difference in <d> between the heating(solid circles) and the cooling (open circles), indicating a reversiblechange without thermal hysteresis.

FIG. 10 b shows the pH independence of the apparent diameter of EPCliposomes. As seen in FIG. 10 b, liposomes were prepared at pH 7.5(initial point, X); pH was increased by addition of 0.2 M NaOH (opencircles) and decreased by addition of 0.1 M HCl (solid circles). SinceEPC is a neutral phospholipid, pH-responsive fusion of its lipid bilayeris not expected. However, DLS revealed a tendency of liposomes toaggregate at low pH (pH<3), a result ascribed to possible protonationand neutralization of the P⁻-N⁺ dipole in phosphocholine on the liposomeinterface and/or to destruction of the hydration shell includinginterfacial water molecules in close contact with lipid polarhead-groups.

Properties of Nanogels

Poly(N-isopropylacrylamide) (PNIPA) andpoly(N-isopropylacrylamide-co-1-vinylimidazole) (PNIPA-VI) nanogels werecharacterized in terms of their response to changes in environmentalconditions. In the following, properties of PNIPA and PNIPA-VI nanogelsare described in §4.2.2.1 and §4.2.2.2, respectively.

Properties of PNIPA Nanogels

After removing solubilized phospholipid and detergent molecules andmixed micelles by dialysis against water and concentrating thissuspension by evaporation in a temperature gradient, resultant PNIPAnanogels have average diameters of around 200 nm (FIG. 11 a),considerably smaller than that for the nanogels in solution containingdetergent and lipid molecules (cf. FIG. 12 b). This finding indicatesthat PNIPA hydrogel particles in the surfactant solution can accommodatesome surfactant molecules on the surface or/and within the gel network,e.g., in the form of micelles.

Since PNIPA gels exhibit a volume phase transition temperature atT_(V)˜32° C., increasing the temperature above T_(V) is expected tocause shrinking of the PNIPA hydrogel particles. Interestingly, at 35°C. (FIG. 12 c) the peak position for the larger particles shiftedtowards smaller sizes whereas at 40° C. (FIG. 12 d) the peak moved tolarger sizes. Herein, the absolute intensities of light scattered fromsmall particles (2) did not change but the absolute scatteringintensities of large particles progressively increased upon elevatingthe temperature. The latter behavior can be explained only if shrunkenPNIPA hydrogel particles aggregate.

Properties of PNIPA-VI Nanogels

Fabricating hydrogel particles using liposomes as reactors was confirmedby removing the vesicle phospholipid bilayer from the lipobeads withdetergent (T_(X-100)). As shown in FIG. 7 b two peaks corresponding tothe mixed detergent/lipid micelles (˜9 nm) and the PNIPA-VI nanogels(˜150 nm) appeared at pH 7.5 and 25° C. The pH was reduced to 2.5 todetermine if the ionizable VI monomers were incorporated within thePNIPA network. The acidic environment should result in ≡NH⁺Cl⁻ groups inthe gel particles. FIG. 7 c shows that the sizes of PNIPA-VI hydrogelparticles at pH 2.5 increased (˜250 nm) compared with those of the gelparticles with the ═N— groups at pH 7.5. The estimated swelling ratio ofα≈4.6 demonstrates the pH-sensitivity of the prepared PNIPA-VI nanogels.

Properties of Lipobeads

Lipobeads combine the properties of liposomes and the nanogels theyenclose to create a system that is sensitive to changes in environmentalconditions. In the following, unanchored PNIPA lipobeads are describedin §4.2.3.1 and anchored PNIPA-VI lipobeads are described in §4.2.3.2.

Properties of Unanchored PNIPA Lipobeads

PNIPA lipobeads were prepared as described in §4.1.3. FIG. 12 a showsthat a relatively broad size distribution of PNIPA lipobeads with a peakat around 250 nm are obtained after UV exposure of a diluted LUVsuspension containing initial components of PNIPA gels (See FIG. 13,line 3). FIG. 12 b demonstrates that again, addition of T_(X-100) in amolar ratio of 45:1 (detergent/lipid) results in two peaks with maximaat 9 nm and 250 nm ascribed to the mixed detergent-phospholipid micellesand the PNIPA hydrogel particles, respectively. The ratio betweenscattering intensities of small and large particles indicates that theconcentration of nanogels is relatively low.

Properties of Anchored PNIPA-VI Lipobeads

In contrast to PNIPA lipobeads, the average size of the anchoredPNIPA-VI lipobeads did not change during volume phase transition (Seelarge peak in FIG. 4 d). Moreover, only a 3% increase in the totalscattering intensity indicated that the aggregation of the anchoredlipobeads was weak at elevated temperatures. After cooling (FIG. 4 c),the size of the anchored lipobeads was entirely restored. The observedreversibility of the swelling/deswelling behavior indicates that theanchored lipobeads aggregate without fusion. In other words, they toucheach other but do not form a “giant” lipobead. The aggregates ofanchored lipobeads shown in FIG. 4 d also disassemble more easily thanthe “giant” lipobeads.

Anchored PNIPA-VI lipobeads also showed a high stability againsttemperature and pH changes (See FIGS. 14 a and 14 b, respectively). Itis likely that PNIPA-VI nanogels shrink at T_(V)˜37° C. (See FIG. 15) ormay exhibit swelling/deswelling from pH 7 to pH 2 (See FIG. 16).However, such changes of the gel size are hidden by the temperature orpH stabilities of the lipid bilayer since the PNIPA-VI nanogels areinside the liposomes. Another reason for the pH-independence of anchoredlipobeads could be that the hydrophobic chains of the anchors penetratethe bilayer, which acts as a barrier to pH changes. Thus the pH of theliposome's interior and exterior may be different.

Properties of Giant Lipobeads

Collapsing temperature-sensitive hydrogels at temperatures above T_(V)causes fast nanogel aggregation. Data confirmed that lipobeadscontaining temperature-sensitive hydrogels also aggregate quickly attemperatures above T_(V). During aggregation, fast fusion of thephospholipid bilayer also occurs if the bilayer is not stabilized bynanogels' anchors. Giant lipobead formation appeared to be irreversible.This may be due to the giant lipobeads having minimal free energy incomparison with separated and aggregated lipobeads. FIG. 3 c shows thesize distribution curve for PNIPA giant lipobeads after cooling to 25°C. and allowing the temperature to equilibrate for 2 hours. Theresulting two peaks indicate that a portion of the giant lipobeads didnot break up into elementary lipobeads.

CONCLUSIONS

As can be appreciated by the foregoing, the present invention can beused to produce lipobeads. Since such assemblies combine the propertiesof nanogels and liposomes, the present invention provides a system thatcan respond quickly to environmental changes and therefore opens up manynew potential applications for lipobeads.

1. A method for producing an anchored lipobead defined by a hydrogel,having a diameter of less than 1 μm, encapsulated in a lipid bilayer,the method comprising: a) encapsulating hydrogel-forming components intoliposomes, wherein the hydrogel-forming components include initiator,cross-linker, and polymer-forming monomers; b) encapsulatinganchor-forming components into liposomal lipid bilayers, wherein theanchor-forming components include water-insoluble hydrophobic monomers,and c) co-polymerizing the polymer-forming monomers of thehydrogel-forming components and hydrophobic monomers of theanchor-forming components, thereby forming lipobeads.
 2. The method ofclaim 1 further comprising: d) diluting a large unilamellar vesicles(LUV) suspension before polymerization to prevent polymerization outsidethe liposomes.
 3. The method of claim 1 wherein the polymer-formingmonomers contain a vinyl group.
 4. The method of claim 1 wherein thepolymer-forming monomers are selected from a group consisting ofacrylamide, N-isopropylacrylamide,N-isopropylacrylamide-co-1-vinylimidazole, N,N-dimethylacrylamide,N,N-diethylacrylamide, 1-vinylimidazole, sodium acrylate, sodiummethacrylate, 2-hydroxyethylmethacrylate (HEMA), N,N-dimethylaminoethylmethacrylate (DMAEMA), N-[tris(hydroxymethyl)methyl]acrylamide,1-(3-methacryloxy)propylsulfonic acid (sodium salt), allylamine,N-acryloxysuccinimide, N-vinylcaprolactam, 1-vinyl-2-pyrrolidone,2-acrylamido-2-methyl-1-propanesulfonic acid (sodium salt),(3-acrylamidopropyl)trimethylammonium chloride, anddiallyldimethylammonium chloride.
 5. The method of claim 1 wherein theliposome is selected from group consisting of egg yolkL-α-phosphatidylcholine (EPC),1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC),1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC),1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC),1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC),1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC),1,2-dioleoyl-sn-glycero-3-phosphaethanolamine (DOPE),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphaethanolamine (POPE),1,2-dimyristoyl-sn-glycero-3-phosphaethanolamine (DMPE),1,2-dipalmitoyl-sn-glycero-3-phosphaethanolamine (DPPE), and1,2-distearoyl-sn-glycero-3-phospharthanolamine (DSPE).
 6. The method ofclaim 1 wherein the cross-linker is selected from a group consisting ofN,N′-methylenebisacrylamide, N,N′-(1,2-dihydroxyethylene)bisacrylamide,ethylene glycol diacrylate, di(ethylene glycol) diacrylate, tri(ethyleneglycol) diacrylate, tetra(ethylene glycol) diacrylate, ethylene glycoldimethacrylate, di(ethylene glycol) dimethacrylate, tri(ethylene glycol)dimethacrylate, tetra(ethylene glycol) dimethacrylate, andpentaerythritol triacrylate.
 7. The method of claim 1 whereinphotopolymerization is accomplished by a photoinitiator, and wherein thephotoinitiator is selected from a group consisting of2,2-diethoxyacetophenone, 2,2-dimethoxy-2-phenylacetophenone (IRGACURE651), 1-[4-(2-hydroxyethoxy)phenyl]-2-hydroxy-2-methyl-1-propan-1-one(IRGACURE 2959), 2-hydroxy-2-methylpropiophenone, and2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone.
 8. The method ofclaim 1 wherein redox polymerization is accomplished by a redoxinitiator, and wherein the redox initiator is selected from a groupconsisting of ammonium persulfate, potassium persulfate,2,2′-azobis[2-methyl-N-(2-hydroxyethyl)propionamide (VA-086),2,2′-azobis(2-amidinopropane)dihydrochloride (V-50),4,4′-azobis(4-cyanovaleric acid).
 9. The method of claim 1 wherein theanchor-forming components are N-alkylacrylamides.
 10. The method ofclaim 9 wherein the N-alkylacrylamides are selected from a groupconsisting of N-octadecylacrylamide, N-dodecylacrylamide, andN-octylacrylamide.
 11. The method of claim 1 wherein the hydrogel has adiameter of approximately 30 nm to approximately 1000 nm.
 12. The methodof claim 1 wherein the lipobead generated has a diameter ofapproximately 30 nm to approximately 1000 nm.
 13. A method for producinga giant lipobead defined by an aggregated plurality of hydrogelparticles, each having a diameter less than 1 μm, encapsulated in alipid bilayer, the method comprising incubating a solution of anchoredor unanchored lipobeads for more at least one month, thereby aggregatinglipobeads and forming giant lipobeads.
 14. The method of claim 13wherein lipobeads are incubated at temperatures below the volume phasetransition temperature of the hydrogel particles.
 15. The method ofclaim 13 wherein the hydrogel particles encapsulated within theliposomes do not shrink.
 16. The method of claim 13 wherein thelipobeads have diameters of approximately 30 nm to approximately 1000nm.
 17. The method of claim 13 wherein the giant lipobead generated hasa diameter of approximately 100 nm to approximately 3000 nm.
 18. Themethod of claim 13 wherein the hydrogel particles are formed frommonomers selected from a group consisting of acrylamide,N-isopropylacrylamide, N-isopropylacrylamide-co-1-vinylimidazole,N,N-dimethylacrylamide, N,N-diethylacrylamide, 1-vinylimidazole, sodiumacrylate, sodium methacrylate, 2-hydroxyethylmethacrylate (HEMA),N,N-dimethylaminoethyl methacrylate (DMAEMA),N-[tris(hydroxymethyl)methyl]acrylamide,1-(3-methacryloxy)propylsulfonic acid (sodium salt), allylamine,N-acryloxysuccinimide, N-vinylcaprolactam, 1-vinyl-2-pyrrolidone,2-acrylamido-2-methyl-1-propanesulfonic acid (sodium salt),(3-acrylamidopropyl)trimethylammonium chloride, anddiallyldimethylammonium chloride.
 19. The method of claim 13 wherein thelipid bilayer is selected from group consisting of egg yolkL-α-phosphatidylcholine (EPC),1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC),1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC),1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC),1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC),1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC),1,2-dioleoyl-sn-glycero-3-phosphaethanolamine (DOPE),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphaethanolamine (POPE),1,2-dimyristoyl-sn-glycero-3-phosphaethanolamine (DMPE),1,2-dipalmitoyl-sn-glycero-3-phosphaethanolamine (DPPE), and1,2-distearoyl-sn-glycero-3-phospharthanolamine (DSPE).
 20. The methodof claim 13 wherein the giant lipobead is a combined giant lipobead. 21.The method of claim 20 wherein the combined giant lipobead containshydrogels loaded with different compartments.
 22. The method of claim 20wherein the combined giant lipobead contains hydrogels filled withdifferent liquid media.
 23. The method of claim 20 wherein the combinedgiant lipobead contains hydrogels functionalized with different ligands.24. The method of claim 20 wherein the combined giant lipobead generatedhas a diameter of approximately 100 nm to approximately 3000 nm.